Noi/NOTES/2014-3-29: Difference between revisions

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Line 235: Line 235:
  -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
  -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
===== Modified BSPP capture =====
===== Modified BSPP capture =====
'''Program'''
'''Program'''
  -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h  
  -> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h  
  -> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP)
  -> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP)

Revision as of 05:27, 1 April 2014

DNA methylation sensitivity test assay - MONOD Project, MONOD_GP1_V4 probe set (BSPP capture)

[Link to calendar]

  • Continued from: [[1]]
File:MONOD-sensitivity-test-plans.png
- For all bis-DNA input, I will use the same concentration of probe in capture reaction by using 500:1 probe:target ratio for 300ng bis-DNA

BSPP capture set up

Probe calculation

Probe:target 500:1 Unit Calculation
Probe size (number of individual probe in oligo mix) 7,110 probes
Human bis-cvt gDNA required 300 ng
Human gDNA MW 1.822E+12 g/mole 3.0E+09 * 607.4Da/bp
Human gDNA( 300ng) 1.646E-19 mole 300E-09g / 1.822E+12g/mole
Probe required (500:1) 8.232E-17 mole 500 * 1.098E-19mole
Probe mix MW (size 7,110probes, 110nt) 2.381E+08 g/mole 7,110 probes * (110bp *303.7Da/bp+79Da)
Mass of probe required 1.960E-08 g 8.232E-17mole * 2.381E+08g/mole
Mass of probe required 19.60 ng
Probe conc 6.87 ng/ul
Volume of probe 2.85 ul

Two parts of experiment

  1. Low-magnitude amplification before BSPP capture
  2. No amplification before capture

1) Low-magnitude amplification before BSPP capture

Sampe description Tube IDs
5ng_STD BSPP capture 5a-S
10ng_STD BSPP capture 10a-S
25ng_STD BSPP capture 25a-S
50ng_STD BSPP capture 50a-S
5ng_modified BSPP capture 5a-M
10ng_modified BSPP capture 10a-M
25ng_modified BSPP capture 25a-M
50ng_modified BSPP capture 50a-M
NTC NTCa-S

- Dilute bis-JURKAT 38.3ng/ul --> 2.5, 5.0, 25 ng/ul

Stock 38.3ng/ul H2O Total volume (ul) Final conc. (ng/ul)
6.53 93.47 100.00 2.50
13.05 86.95 100.00 5.00
32.64 17.36 50.00 25.00

- Mix bis-JURKAT with Ampligase buffer and 2uM N9 random primers

bis-JURKAT Conc. (ng/ul) Amount required (ng) Volume forXXng (ul) bis-JURKAT H2O
5a-S 2.50 5.00 2.00 5a-S 4.30
10a-S 5.00 10.00 2.00 10a-S 4.30
25a-S 5.00 25.00 5.00 25a-S 1.30
50a-S 25.00 50.00 2.00 50a-S 4.30
5a-M 2.50 5.00 2.00 5a-M 4.30
10a-M 5.00 10.00 2.00 10a-M 4.30
25a-M 5.00 25.00 5.00 25a-M 1.30
50a-M 25.00 50.00 2.00 50a-M 4.30
NTCa-S 0.00 0.00 0.00 NTCa-S 6.30
Mix I: Ampligase buffer & N9 primer mix
N9 & Buffer Mix 1x rxn 12x rxn
10X AmpLigase Buffer 1.00 12.00
100uM N9 0.20 2.40
Total 1.20
Mix II: Klenow, exo- & dNTP mix
Klenow & dNTP mix 1x rxn 12x rxn
10mM dNTP Mix 0.20 2.40
Klenow Fragment, exo - (5U/ul) 0.50 6.00
H2O 1.80 21.60
Total 2.50
Mix III: MONOD_GP1_V4 BSPP & Ampligase buffer mix
Probe & Buffer Mix 1x rxn 9.5x rxn
10X AmpLigase Buffer 1.00 9.50
MONOD_GP1_V4 probe (19.60ng) 2.85 27.08
H2O 6.15 58.43
Total 10.00
- Add 1.2ul of Ampligase/N9 primer mix to bis-JURKAT
- 94C, 3 min --> 25C 3min
- Add 2.5ul of Klenow Fragment & dNTP mix (I moved the tube from thermocycler and mix samples at RT)
- 25C, 1h --> 75C, 20min
- Add 10ul of probe & buffer mix
- Layer capture tubes with 2 drops of mineral oil
- Place the tube on two thermocyclers, standard capture protocol and multiple rounds of circularization protocol.

2) No amplification before BSPP capture

Samples Code
5ng_STD BSPP capture 5-S
10ng_STD BSPP capture 10-S
25ng_STD BSPP capture 25-S
50ng_STD BSPP capture 50-S
100ng_STD BSPP capture 100-S
200ng_STD BSPP capture 200-S
300ng_STD BSPP capture 300-S
5ng_modified BSPP capture 5-M
10ng_modified BSPP capture 10-M
25ng_modified BSPP capture 25-M
50ng_modified BSPP capture 50-M
NTC NTC-S
Capture set up
bis-JURKAT Conc. (ng/ul) Amount required (ng) Volume forXXng (ul) bis-JURKAT H2O (ul) MONOD_GP1_V4
probe (19.60ng)
10X AmpLigase
Buffer
Total (ul)
5-S 2.50 5.00 2.00 5-S 13.15 2.85 2.00 20.00
10-S 5.00 10.00 2.00 10-S 13.15 2.85 2.00 20.00
25-S 5.00 25.00 5.00 25-S 10.15 2.85 2.00 20.00
50-S 25.00 50.00 2.00 50-S 13.15 2.85 2.00 20.00
100-S 38.30 100.00 2.61 100-S 12.54 2.85 2.00 20.00
200-S 38.30 200.00 5.22 200-S 9.93 2.85 2.00 20.00
300-S 38.30 300.00 7.83 300-S 7.32 2.85 2.00 20.00
5-M 2.50 5.00 2.00 5-M 13.15 2.85 2.00 20.00
10-M 5.00 10.00 2.00 10-M 13.15 2.85 2.00 20.00
25-M 5.00 25.00 5.00 25-M 10.15 2.85 2.00 20.00
50-M 25.00 50.00 2.00 50-M 13.15 2.85 2.00 20.00
NTC-S 0.00 0.00 0.00 NTC-S 15.15 2.85 2.00 20.00
Probe & AmpLigase Buffer mix
Components Volume (ul) 12.5x
10X AmpLigase Buffer 2.00 25.00
MONOD_GP1-V4 (6.87 ng/ul) 2.85 35.63
Total 4.85
Split samples into two groups, incubate on thermocycler following standard BSPP capture (s) and modified BSPP capture (multiple round of circularization, m)
Standard BSPP capture
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
Modified BSPP capture
Program
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h 
-> add 2.5ul SLN mix (2U/ul Hemo Klentaq (NEB); 0.5U/ul AmpLigase; 200uM dNTP)
-> 55C 4h
-> 4x of [95C, 1min -> 55C, 4h)
-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.